Lrp5 works through the canonical Wnt/p-catenin signaling pathway during skeletal development. The HBM mutation (G171V) in Lrp5 results in up-regulation of bone mass such that the bones are over adapted to daily loads, but normal in shape and turnover rates. Our general hypothesis is that in the adult skeleton, the LRP5/Wnt pathway is directly involved in mechanical regulation of bone mass such that there is a dose response relationship between LRP5 expression level with mechanical stimulation and that the HBM mutation further increases the response. Our alternate hypothesis is that the HBM mutation increases mechano-sensitivity independent of Lrp5 expression level. The specific hypotheses we will test are: 1) increased LRP5 gene expression, and especially the HBM mutation, increase the bone response to mechanical loading, 2) Lrp5 and the HBM mutation modulate the osteogenic response to mechanical stimulation through the canonical Wnt pathway, 3) while parathyroid hormone (PTH) will be additive with loading, there will be no HBM or LRP5 dose effect with PTH, and 4) prostaglandins are important in the LRP5 (HBM) response to mechanical loading such that suppression will inhibit the anabolic response and addition of PGE2 will stimulate a dose response. To establish a dose response and test our hypotheses we will compare four mouse genotypes with a range of Lrp5 expression: the Lrp5 heterozygous knockout (Lrp5+/-), wild type C57BI/6, the human LRP5tg transgenic, and the HBMtg transgenic with the human WT or G171V mutation inserted. We will use in vitro primary cell cultures to specifically test the Writ canonical pathway and in vivo studies to determine the organ level regulation of bone turnover. The specific aims are to 1) establish the threshold, dose response, and half-maximal effective dose (EDso) for loading in vivo and in vitro; 2) in vitro compare the effects of fluid flow to a Wnt agonist on the Wnt canonical pathway for GSK 3P ser9 phosphorylation, nuclear localization of p-catenin, and response to disruption of the pathway by DKK1 or siRNA for p-catenin; 3) in vivo examine the interaction of loading and PTH 1-34 to determine the specificity of the LRP5 and HBM mutation response to anabolic stimulation; and 4) in vivo combine loading with a Cox2 inhibitor or PGE2, to examine the role of prostaglandins in mediating Lrp5 responses to loading. In vivo studies will examine long term (4 wk) adaptation to treatment with histomorphometry and then acute responses via mRNA and histochemistry (BrDU labeling, apoptosis, p-catenin expression, alkaline phosphatase, and TRAP).The in vitro studies will examine primary osteoblast cell cultures for mRNAand protein expression along with cell proliferation. Both systems will examine genes known to be up regulated by mechanical loading in HBMtg that are Wnt target genes - WISP-2 (Wnt induced secreted protein 2), Wnt related -Cox-2, eNOS, Cyclin-D1, and non-target genes - Lrp5, OPGandWnt 10b. These studies provide a unique opportunity to test the role of LRP5 in mechanical regulation of bone mass through Wnt signaling pathways.